Abstract This application is to renew a grant to study RNA methylation in pneumoviruses. Pneumoviridae is a new virus family, created in 2016 by elevating the paramyxoviral subfamily Pneumovirinae to family status. The Pneumoviridae family includes two medically important pathogens, human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), which are the leading causative agents of acute viral respiratory tract infections in infants, young children, the elderly, and immunocompromised individuals. Despite the enormous economic loss and emotional burden these viruses cause, no vaccines or anti-viral drugs are currently available. Development of such agents requires a better understanding of all aspects of their life cycle. In the last grant period, we have revealed the unique mechanism of mRNA capping and cap methylation in pneumoviruses. We recently discovered that pneumovirus genome, antigenome, and mRNAs are also methylated at internal adenosine residues to form N6-methyladenosine (m6A) by host m6A methyltransferase complex. Although m6A methylation has been discovered in viral RNA in early 1970s, the biological function of m6A in the virus life cycle, pathogenesis, and immunity has been a mystery for four decades. We have found that the internal m6A methylation in viral RNAs promotes pneumovirus replication and gene expression. The objectives of the current application are to determine the roles of internal m6A methylation in pneumovirus replication and pathogenesis in vivo; and to define mechanism(s) by which m6A methylation modulate pneumovirus life cycle. Our Specific Aims are: (1) to define the host m6A machinery that regulates pneumovirus replication and gene expression; (2) to define the mechanism(s) by which internal m6A promote pneumovirus replication and gene expression; and (3) to define the roles of m6A methylation in pneumovirus replication and pathogenesis in a cotton rat model. The successful completion of this work will not only advance our understanding of m6A methylation in regulating pneumovirus life cycle and pathogenesis, but also provide a novel approach for developing live attenuated vaccine candidates and antiviral drugs by inhibiting viral m6A methylation.